Curcumin and its Analogs and Carriers: Potential Therapeutic Strategies for Human Osteosarcoma

Curcumin is a natural polyphenol phytochemical derived from turmeric with antioxidant, anti-inflammatory, and anticancer properties but is concerned about poor solubility in water, absorption, and metabolic stability. Potent metastatic osteosarcoma is the most common primary bone cancer in children, adolescents, and young adults. It is responsible for low survival rates because of its high rate of metastasis to the lungs. To improve poor bioavailability, numerous curcumin analogs were developed to possess anticancer characteristics through a variety of biological pathways involved in cytotoxicity, proliferation, autophagy, sensitizing chemotherapy, and metastases. This review provides an overview of their various pharmacological functions, molecular mechanisms, and therapeutic potential as a remedy for human osteosarcoma. To enhance therapeutic efficacy, several liposomal nanoparticles, nanocarriers, multifunctional micelles, and three-dimensional printed scaffolds have also been developed for the controlled delivery of curcumin targeting human osteosarcoma cells. Consequently, curcumin and several potential analogs and delivery formulations are optimistic candidates to improve the currently available strategy for human osteosarcoma. However, further insight into the mechanism of action of promising curcumin analogs and the development of carriers in clinical trials of osteosarcoma needs to be investigated to improve their overall potency and clinical utility, in particular the anti-metastatic effect.


Introduction
Turmeric (curry powder), a cooking spice, a food preservative, and a natural yellow food coloring in many foods, such as curries and mustards, as well as a cosmetic agent for skin care in Asia, Europe, and America, has been integral to traditional medicinal systems for centuries, such as Indian Ayurveda [1] and Chinese herbs [2]. For thousands of years, turmeric has been used to treat common colds, fevers, stomachaches, liver diseases, open wounds, skin diseases, and chronic inflammations. After epidemiological studies, turmeric may have the ability to lower cancer incidence rates by 10-50% among those who regularly consume the spice [3].

Ivyspring International Publisher
Indian plant Curcuma longa Linn. by two German scientists Vogel and Pelletier in 1815 [5,6]. It is a member of the Zingiberaceae (ginger) family of perennial botanicals with a long history of traditional uses that range from folk medicine to various culinary preparations [7].

Osteogenic sarcoma (osteosarcoma)
Primary malignant bone neoplasms are extremely rare malignancies and account for less than 0.2% of all cancers [26]. Osteosarcoma, the most prevalent primary bone malignancy, accounts for approximately 35% of all malignant bone tumors [27] and 3-5% of pediatric cancers [26]. It shows a peak incidence between 11 and 15 years with an estimated incidence of 3 cases/million population/year and about 6 per million children [28][29][30][31] as well as a second peak incidence in older adulthood and 2 per million adults [31]. In general, 80-90% of osteosarcoma occurs in long bones, especially the metaphysis, and the femur, tibia, and humerus represent approximately 85% of them [28]. Currently, standard clinical procedures for osteosarcoma are preoperative (neoadjuvant) chemotherapy, including cisplatin, doxorubicin (DOX), adriamycin, methotrexate, ifosfamide, and etoposide, en bloc or radical excision of the primary bone tumor or amputation of the diseased limb and complex reconstructions, and adjuvant chemotherapy [32].
Prognostic factors for osteosarcoma patients include patient age, tumor site and size, presence and location of metastasis, chemotherapy response, and surgical type and margins [26,33]. Because osteosarcoma cells are not easily killed by radiation, radiotherapy can sometimes be used in patients with positive surgical margins [26,34]. While radiotherapy is administered to try to kill the remaining cancer cells, adjuvant chemotherapy is often followed. Thus, neoadjuvant chemotherapy is routinely used to treat preoperative occult metastases and preserve the diseased limb in more than 90% of osteosarcoma patients [28,35]. However, most treatment failures and high mortality rates result from its metastatic vigor [36], even when aggressively treated with neoadjuvant chemotherapy. If the necrosis of the resected tumor is less than 90% after neoadjuvant treatment, adjuvant chemotherapy regimens can be altered [37].
Generally, four (osteoblastic, fibroblastic, chondroblastic, and telangiectatic) pathologic subtypes of conventional high-grade human osteosarcoma are clinically identified, while no significant differences in clinical outcome correlate with the various subtypes, including the chondroblastic subtype without a favorable response to chemotherapy [29,31]. To date, the long-term survival rate is 60-75% at 5 years for patients with localized tumors [35], but it is drastically reduced to 20-30% if positive pulmonary metastases at diagnosis [38,39]. Potent lung metastasis remains the leading cause of death in osteosarcoma [30,31]. The development of adjuvant therapies to target the molecular basis for abrogating osteosarcoma cell proliferation, invasion, and migration and improving survival rates is crucial [40].

Molecular carcinogenesis, cell death, and metastasis of osteosarcoma
Carcinogenesis (oncogenesis) is a composite process of transforming normal cells into cancer cells in which multiple cell signaling pathways and hundreds of molecules are dysregulated, including biochemical molecules of growth factors and receptors, transcription factors, enzymes, kinases, cytokines, and genes that regulate proliferation, angiogenesis, and apoptosis [41]. As a result of activating extrinsic (death receptor) and/or intrinsic (mitochondria) caspases, apoptosis is initiated that manifests as cell shrinkage, plasma membrane bleb, chromatin condensation, and nuclear DNA fragmentation [42][43][44]. In the transmission of the apoptotic pathway, stress-inducible molecules, such as MAPKs)/extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, as well as phosphoinositide 3-kinase (PI3K)/Akt are involved [45][46][47].
After carcinogenesis, several distinct hallmarks of cancer cells develop, such as evading apoptotic cell death, endless proliferation, and distant metastasis, resulting in host mortality. During tumor growth or disease, a physiological or pathological process, newly formed vascular vessels evolve from pre-existing vessels through angiogenesis [48]. Autophagy (autophagocytosis), a cellular degradation process, allows cells to remove unnecessary or dysfunctional components by inducing the lysosomal recycling of intracellular nutrients and caspaseindependent cell death in conditions of starvation [49]. The orderly degradation and recycling of cellular components also play an imperative role in the homeostasis of non-starved cells. In addition to the promotion of apoptosis [50], JNK and p38 can induce autophagic cell death to regulate autophagy programs [45,51].
Cancer metastasis involves the dissociation of malignant tumor cells, the epithelial-mesenchymal transition (EMT), intravasation into the circulation, travel through the bloodstream and lymph fluid, survival and evasion of the host immune system, extravasation, mesenchymal-epithelial transition (MET) and adhesion, and colonization and angiogenesis at a distant site [52,53]. Motile cancer cells after EMT degrade the extracellular matrix (ECM) and invade the basement membrane of blood and lymph vessels through a series of discrete steps and various proteases [53]. Mesenchymal-derived sarcoma cells are different from epithelial-derived carcinoma cells and are poorly connected to neighboring cells to embed themselves within the ECM. As traveling tumor cells colonize distant sites, the opposite MET is required for metastasis progression.
However, the concept of mesenchymal-derived osteosarcoma cells in EMT with the capability to initiate the metastasis cascade has been further applied in the past decade [54][55][56][57].
Like other cancer cells, a variety of pathways, including MAPKs, PI3K/Akt, NF-κB, and JAK/STAT3 participate in various signaling cascades of cell motility, adhesion, migration, and invasion in osteosarcoma [47,56,58,59]. Identifying molecular insights into the signal transduction pathways involved in carcinogenesis, induction of cell death, and metastasis of osteosarcoma leads to the development of effective treatments available for patients with or without metastasis [30,31,53]. As a natural product with pleiotropic anticancer activities, curcumin diversifies its inhibitory effects on a multitude of pathways and biochemical cascades involved in tumorigenesis, proliferation and angiogenesis, apoptosis, and metastasis [2]. Figure 1 shows a summary of the structure of curcumin (bis-α,β-unsaturated β-diketone), which is a 1,3-diketone with methoxy and alcohol groups attached to its two terminal benzene rings, and its natural and synthetic analogs involved in the molecular impact on human osteosarcoma cells [60]. The chemical structures of curcumin, DMC, and BDMC are those of a diarylheptanoid with differently substituted aryl rings and a conjugated aliphatic structure. Curcumin is not soluble in water at acidic and neutral pH [8]. Due to interactions between acidic alpha hydrogens and basic molecules, the keto-enol structure of curcumin is formatted in the blood, a cardinal keto pattern in acidic and neutral solutions, and a stable enol pattern in alkaline media [61].

Absorption and excretion of curcumin
Because curcumin contains the symmetry of electronegative oxygen groups, the molecule has low solubility: approximately 11 ng/mL in water to be absorbed, approximately 75% of oral intake at a dose of 1 g/kg/rat is excreted through the stool passage, and negligible amounts are found in urine [62,63], indicating its poor absorption from the gastrointestinal tract ( Figure 2). In another study by the same researchers, 10, 80, and 400 mg/rat of curcumin were administered orally, with a constant percentage (approximately 60-66%) of absorption regardless of the administered dose, and approximately 40% of curcumin in an unchanged form could be found in feces over 5 days. However, no detectable amounts of curcumin have been observed in heart blood and urine, and negligible amounts have been observed in the liver (< 5 μg/mL in portal blood) and kidney after 24 h [64,65]. In a human study, administration of a single dose of 2 g of curcumin orally generated a plasma concentration of fewer than 0.01 μg/mL at 1 h and then a gradual decline to zero at 3 h [66], also indicating poor intestinal absorption.
Oral administration of 4-8 g of curcumin at 1-2 h in humans could obtain maximum plasma levels of as little as 0.51-1.77 μM and gradually decrease in 12 hours through avid intestinal sulfation, glucuronidation, and reduction [67]. In a human clinical trial, an oral intake of 3.6 g of curcumin at 1 h could produce 11.1 nM of curcumin in plasma [68]. After administration of 10 and 12 g of curcumin orally in healthy human volunteers, its glucuronide and sulfate conjugates could be detected mainly in plasma [69,70], and they appeared to offer low activity, low cell permeability, and a short plasma half-life compared to the free form [68,71]. When administered orally at a dose of 500 mg/kg in rats, 1% bioavailability of curcumin with the parent drug at levels near the detection limit between 5 and 10 nM has been detected in the plasma [72,73]. In another rat study, a low plasma level of 15 ng/mL of curcumin after oral administration of 1 g/kg curcumin for 50 minutes again suggests poor intestinal absorption of curcumin [74].
Not through intestinal absorption, but transport by mesenteric and extra-mesenteric, portal and extra-portal, or lymphatic vessels, intra-peritoneal (i.p.) injection of 0.1 g/kg curcumin to mice at 15 min partially subjected to the elimination of liver first-pass could yield a maximal concentration of 2.25 μg/mL of curcumin in plasma and a maximal amount of curcumin (117 μg/g) in the intestine at 1 h [75]. Using intravenous (i.v.) injections directly transported into the circulation, 40 mg/kg of curcumin by i.v. injection in rats decreased in plasma in 1 h [73]. After i.v. administration of 10 mg/kg of curcumin in rats, the maximum concentration of unchanged curcumin appeared 0.36 μg/mL in plasma, while, after oral administration of a 50-fold higher dose of curcumin (500 mg/kg), only 0.06 μg/mL of curcumin could be present in serum and the time to reach the maximum concentration was 41.7 min [72].

Metabolism and distribution of curcumin
After absorption, curcumin is subjected to conjugations such as glucuronidation and sulfation, as well as reduction at various tissue sites [76,77]. Several studies in rodents and humans have shown that the main organ responsible for the metabolism of curcumin is the liver [78,79]. In the intestinal tract and during its first passage through the liver of humans and rats, curcumin is biotransformed differently from curcumin glucuronide and curcumin sulfate and it is reduced by different reductases to dihydrocurcumin (DHC), tetrahydrocurcumin (THC), and hexahydrocurcumin (HHC), and ultimately catalyzed reduction to octahydrocurcumin (OHC), which is alternatively called hexahydrocurcuminol [75,80,81]. The metabolic reaction of curcumin is carried out extensively in hepatocytes and enterocytes, and then the reductive metabolites are easily conjugated [73,[80][81][82][83]. After first-pass and circulation metabolism, biliary metabolites are the major glucuronides of THC and HHC and the minor dihydroferulic acid with traces of ferulic acid [84]. Due to its poor absorption from the gastrointestinal tract, approximately 75% of the oral intake of curcumin is excreted through the stool passage and negligible amounts are found in the urine. In the intestinal tract and during its first passage through the liver, curcumin is conjugated differently to the glucuronide and sulfate of curcumin in the intestinal tract and liver. Curcumin with or without conjugation is also reduced to dihydrocurcumin (DHC), tetrahydrocurcumin (THC), and hexahydrocurcumin (HHC), and ultimately catalyzed reduction to octahydrocurcumin (OHC), which is alternatively called hexahydrocurcuminol. After first-pass and circulation metabolism, the biliary metabolites are the main glucuronides of THC and HHC and the minor dihydroferulic acid with traces of ferulic acid. Not through intestinal absorption, intraperitoneal (i.p.) injection of curcumin partially subjects to hepatic first-pass elimination, while intravenous (i.v.) injections are directly transported into the circulation.
The metabolic conjugation of curcumin with activated sulfuric or glucuronic acids is more abundant in human intestinal fractions than in rat ones; however, conjugation in human hepatic fractions is less extensive than in rat liver tissues. While curcumin glucuronides, THC, and HHC metabolites, made primarily in the intestines, have their own pleomorphic anticancer, anti-inflammatory, and antioxidant properties, they are less potent than the parent curcumin [80,85]. Conversely, other studies suggested that DMC, curcuminoid glucuronides, and THC are more active than curcumin itself [86][87][88]. Extensive intestinal and liver metabolic biotransformation of conjugation and reduction of curcumin leads to a reduction in unmetabolized curcumin exposed to the general blood circulation and tissues such as bone other than the digestive tract.
When curcumin circulates in the body, quick alterations in its chemical structure through keto-enol tautomerism (enolization) result in low systemic distribution [89]. The fast elimination of curcumin from the body is another major cause of its short half-life and low biological activity, especially the intestinal and first-pass metabolism of conjugation and reduction. After i.p. and i.v. injections in rodents, curcumin circulates in the body and large amounts of curcumin and metabolites circulating in the body are excreted in bile; however, major metabolites THC and HHC glucuronides may be reabsorbed into the enterohepatic circulation [64]. More than 50% of curcumin is metabolized and secreted into the bile within 2 hours after i.v. administration. Within 72 h after i.p. administration, approximately 73% of curcumin metabolites are excreted in feces and 11% in urine [84]; it indicates that their large elimination occurs through bile with relatively little remaining in tissues. Similarly, 6 h after curcumin i.v. injection, approximately 85% of curcumin metabolites have been found in the bile. In addition to traces of the unchanged drug in the liver and kidney, a trace amount (< 5 μg/mL) in the portal blood has been observed from 15 min to 24 h after oral administration of 400 mg of curcumin to rats, while no curcumin could be detected in the heart blood [65].
In rodents, poor diet bioavailability and curcumin levels found in sera are due in part to avid intestinal and first-pass hepatic metabolic biotransformation [61]. At an oral dose of 2 g/kg in rats, absorption and elimination half-lives of 0.31 ± 0.07 and 1.7 ± 0.5 h have been reported, respectively, and a maximum concentration of 1.35 ± 0.23 μg/mL in sera has been found at 0.83 h [66]. However, at a lower oral dose of curcumin of 1 g/kg in rats, an elimination half-life value of 1.45 h was detected and it showed an insignificant difference from a higher dose of curcumin, indicating the dose independence of the elimination half-life of curcumin in rats [90]. The same dose of 2 g/kg of curcumin has shown undetectable or extremely low serum levels (0.006 ± 0.005 μg/mL) at 1 h in humans [66]. In another study of rats, the elimination half-life values for oral curcumin (500 mg/kg) and i.v. (10 mg/kg) have been detected to be 28.1 ± 5.6 and 44.5 ± 7.5 h, respectively [72], further suggesting poor absorption and rapid metabolism of oral curcumin.
In a clinical pilot study, colorectal cancer patients with liver metastases who received an oral dose of 0.45 to 3.6 g/day curcumin for 1 week could obtain low nanomolar levels of the parent compound and its conjugates in the portal and peripheral circulation [79]. Although trace levels of curcumin products and their metabolic reduction have been detected, no curcumin could be found in liver tissue. Therapeutic doses of curcumin for the liver and other tissues will not be sufficient in humans. In another study, the low bioavailability of oral curcumin has also been observed in patients with advanced colorectal cancer refractory to standard treatments [71]. Similarly, other studies have shown the absence of carcinogenesis inhibition of curcumin against lung and breast tumors due to its low bioavailability [10]. Therefore, the aqueous insolubility, poor absorption, rapid metabolism, and short half-life of curcumin pessimistically hinder its clinical application.
Curcumin and its metabolites have been interestingly observed to affect the gut microbiota [91,92]. These metabolites influence curcumin biotransformation, producing 23 different metabolites and various metabolic pathways, such as acetylation, demethylation, hydroxylation, and reduction [93,94]. The bidirectional interaction between curcumin and the gut microbiota and vice versa suggests the paradox between the poor bioavailability of curcumin and its diverse pharmacological effects. However, curcumin metabolites specifically modulate the microbiota, host intestinal signals, and gut-associated diseases through fine-tuned mechanisms, such as the gut-liver axis and the gut-brain axis [95][96][97][98][99][100][101], while in vivo human studies on the subject are still lacking [102].

Toxicity and tolerance of curcumin
Like reports of various antitumor properties of melatonin and zoledronate depending on different cancer types and cell lines specifically [30,31,53], several signaling mechanisms with specific cell types and different concentrations have been demonstrated for various effects of curcumin in osteosarcoma and other types of cancer cells [45,103]. Curcumin may exhibit highly pleiotropic effects through multiple cascades serially or in parallel on cells and concentrations of curcumin are dependent. For example, curcumin at a low concentration (up to 25 μM) induces osteoblast apoptosis, whereas osteoblast necrosis has been detected when the curcumin concentration increases to 200 μM [104]. However, curcumin at concentrations of 5-25 μM could induce higher cell death in human osteosarcoma MG-63 cells than in human osteoblasts, as malignant cells are much more sensitive to curcumin than their normal cells [19,105].
The higher curcumin uptake rate in cancer cells than in normal cells partially contributes to the differential effects of curcumin on cancer cells versus their normal counterparts [106]. Reduced levels of glutathione in cancer cells that improve curcumin sensitivity may be another factor that contributes to targeted activity [107]. Curcumin suppresses cancer cell proliferation and survival by inhibiting NF-κB but not expressing it in normal cells [108]. Although higher concentrations of curcumin are hepatocytotoxic, small concentrations can protect hepatocytes [19,107]. Furthermore, colon cancer cells are much more sensitive to low doses of curcumin than human dermal fibroblasts, and there may be a degree of selectivity for neoplastic cells [109]. Curcumin induces dose-dependent damage to both the nuclear and mitochondrial genomes, and mitochondrial damage is more extensive in human hepatoma G2 cells [110]. Despite the lack of DNA damage at low doses, curcumin at high doses leads to oxidative stress and damaged DNA, which influences carcinogenesis and causes cancer cell death. However, few adverse events due to curcumin and dose-related toxicity could occur, even at a single oral dose of 12 g and up to 12 g per day for 3 months in humans [61,67].
In addition to the discernible toxicity of mild diarrhea, detectable levels of the parent compound and its conjugates that can inhibit the production of prostaglandin E2 in leukocytes could be generated in plasma and urine after oral doses of 0.5-3.6 g/day curcumin for up to 4 months [68]. It recommends a dose of 3.6 g/day orally for evaluation in clinical trials. Owing to the low nonspecific toxicity to normal cells, the protective and therapeutic effects of curcumin against cancers at almost all stages of carcinogenesis, cell proliferation, and metastasis have received significant attention [111]. Despite excellent tolerance, the widespread clinical application of curcumin has been severely curtailed because of its imperfect aqueous solubility (11 ng/mL), poor absorption, profligate metabolism, and systemic elimination. Extremely low levels (22-41 ng/mL) of curcumin could be present in the serum even after high amounts (8 g/day) of oral intake, indicating its limited circulation and tissue distribution [69].

Analogues and carriers to improve bioavailability
Poor aqueous solubility, low intestinal absorption, enolization, and fast metabolic biotransformation of curcumin result in an inability to reach required blood and tissue concentrations. In the past decade, chemical approaches to solving this problem have been used by making the molecule more polar, more asymmetrical, and/or adding more electronegative groups. In addition, they have simplified the monocarbonyl structure to increase the solubility of curcumin analogs [89,112]. The lack of alpha hydrogen molecules could prevent enolization in the body, causing a delay in the metabolism and elimination of curcumin analogs.
By conjugating with macromolecules and encapsulating in liposomes, micelles, and nanoformulations, several nanoparticles (NPs)-based delivery systems have emerged to enhance water solubility, and absorption, thus ultimate bioavailability, and targeted delivery to promote anticancer effects in osteosarcoma [113][114][115]. Our state-of-the-art review from the PubMed database of inception dates until January 1, 2023, explored novel insights into curcumin's intracellular and molecular mechanisms, as well as its potential analogs in human osteosarcoma cells in vitro and in vivo. Additionally, we compiled the available literature on curcumin in numerous formulations targeting human osteosarcoma.

Curcumin in human osteosarcoma
Curcumin aims at multiple chemotherapeutic pathways and has the tolerability to act as a potential agent for the primary or adjunct treatment of osteosarcoma [116,117] (Figure 3). In seven human osteosarcoma cell lines (U2OS, Saos-2, LM5, Hu09 WT, Hu09 m132, MG-63 WT, and MG-63 M8), curcumin has exhibited dose-dependent growth inhibition with half-maximal growth inhibitory concentrations (IC50) ranging from 14.4 to 24.6 μM [116] (Table 1). Curcumin could induce apoptosis and G 2 /M phase arrest in seven cell lines through the apoptotic signaling pathways, including cleavage of caspase 3 and poly(adenosine diphosphate-ribosyl) polymerase (PARP), a decrease in cellular levels of the anti-apoptotic protein B cell leukemia/lymphoma-2 (Bcl-2), and an increase in cellular levels of the apoptotic protein Bcl-2-associated X protein (Bax).   [121] Down-regulates 14-3-3ɛ expression and alters co-located apoptosis-associated proteins MG-63 7.5 μg/mL Luo Z, et al. 2018 [132] Suppresses proliferation and promotes apoptosis by targeting ITPR1 U2OS 15 μM Possibly suppresses migration and invasion by targeting ITPR1 Sun Y, et al. 2019 [59] Inhibits The migration activities of Saos-2 and U2OS cells have been half-maximally inhibited with 12.5 μM and 25 μM curcumin, respectively, and part detachment of cells with a rounded appearance and almost complete blockage of migration have been achieved at 50 μM curcumin in the seven cell lines. However, the authors could not speculate on the antimigratory potential wildly, because the cytostatic concentration range of 12.5 μM and 25 μM of curcumin could cause cell death to mislead antimigratory information [116].

Curcumin in MG-63 cells
At the right concentrations of 5-25 μM, curcumin could selectively kill human osteosarcoma MG-63 cells with an apoptotic peak in sub-G 1 and changes in nuclear matrix proteins (NMPs) over healthy bone cells [105,118]. NMPs are a class of tissue-specific proteins that modulate signal transduction, gene expression, and apoptosis. 14-3-3ɛ, an NMP, can integrate various signaling pathways by interacting with different proteins to regulate the cell cycle and apoptosis [119,120], and curcumin could suppress the expression level of 14-3-3ɛ and change the co-located apoptosis-associated proteins, including Bcl-2 and Bax, p53, and c-Fos transcription factor in MG-63 cells [121].
Although a low concentration (10 μΜ) of curcumin has reduced the reactive oxygen species (ROS) level in MG-63 cells, a high concentration (80 μΜ) of curcumin could increase the ROS content, the Cyto C release, and the caspase 3 activation [122]. Because the ROS scavenger N-acetyl-L-cysteine (NAC) inhibits the release and activation of the apoptosis protein, ultimately hindering curcumininduced apoptosis, NAC attenuates the toxicity of curcumin, indicating that ROS may contribute to Cyt C release and cell apoptosis in curcumin treatment, indicating curcumin induction of MG-63 cell apoptosis through the ROS/Cyt-C/Caspase-3 pathway.
In MG-63 cells, curcumin inhibits proliferation in the microculture tetrazolium colorimetric (MTT) assay and decreases the expression levels of the microRNA (miRNA/miR)-138 target genes Smad4, NF-κB p65, and cyclin D3 [125]. While overexpression of hsa-miR-138 decreases the Smad4, NF-κB p65, and cyclin D3 expression levels, inhibition of hsa-miR-138 increases them in MG-63 cells, suggesting that curcumin increases the expression of hsa-miR-138 to suppress cell proliferation and invasive capacity by inhibiting its target genes. Regardless, the author's conclusion that 20 μM of curcumin reduced invasive capacity is doubtful, because the curcumin concentration treated at 24 h also showed the potential for cytotoxicity against MG-63 cells (p < 0.05), which is mentioned in the study results [125].
Upon exposure to hypoxic conditions, MG-63 cells have shown increased proliferation and increased invasiveness, and these effects could be prevented with curcumin by inhibiting the up-regulation of Notch 1 induced by hypoxia [126]. While overexpression of Notch 1 through Notch 1 cDNA transfection has ameliorated curcumin-inhibited MG-63 cell growth under hypoxia, the inhibitory effect on MG-63 cell growth in hypoxia is demonstrated by suppressing Notch 1 expression. Based on data from pancreatic duct adenocarcinoma and gastric cancer cells under hypoxia that exhibit greater invasiveness and metastasis [127,128], the authors speculated that curcumin at 5 and 10 μM could inhibit MG-63 cell invasion under hypoxic conditions. However, hypoxic concentrations that could exhibit a cytotoxic effect in the study should be excluded [126].
Increases in apoptosis and accumulation in the G2/M phase of MG-63 cells have been demonstrated after curcumin (10 and 20 μM) treatment by suppressing the p-JAK2/p-STAT3 signaling pathway, and 27.6 μM is its half-maximal inhibitory concentration at 24 h [59]. The authors concluded that curcumin inhibited migration and invasion using the same concentrations of 10 and 20 μM, which could partially contribute to the cytotoxic effect. Hence, the authors should rule out this possibility. Furthermore, in 80 osteosarcoma patients, the mean expressions of p-JAK2 and STAT3 in primary osteosarcoma tissues with lung metastasis (9.23 and 14.82, respectively) are much higher than those without lung metastasis (2.43 and 5.06, respectively), and a positive correlation is evident between both. In MG-63 xenograft mice, curcumin decreases tumor size and p-JAK2 and STAT3 expressions, further validating tumor growth suppression through the JAK2/STAT3 pathway.

Curcumin in U2OS cells
In U2OS cells, curcumin at a concentration of 50 μM has shown the ability to inhibit nicotine-induced ERK expression [129]. Although curcumin and DOX administered together have not shown an additive effect in U2OS cells, additive cytotoxicity with curcumin and cisplatin could be observed [116]. Curcumin induces G1 phase accumulation and undergoes apoptosis (a sub-G 1 peak at 6 h and a G 1 block peak at 12 h) in U2OS cells [130]. Through the increase in mitochondrial membrane permeability by down-regulation of Bcl-2 but drastically up-regulating Bax, Bcl-2 antagonist killer 1 (Bak), and phosphorylated Bcl-2-associated agonist of cell death (p-Bad), curcumin triggers the release of cytochrome c (Cyt C) from mitochondria into the cytosol and the activation of downstream caspase 3 and the appearance of apoptosis.
In addition, curcumin improves the cytotoxicity of Adriamycin in multi-drug resistant cells of U2OS/ADM (human osteosarcoma cell line model) and the accumulation of Rhodamine 123, indicating the reverse mechanism of blocking the function of P-gp in the cellular membrane of U2OS/ADM [131]. Targeting inositol 1,4,5-triphosphate receptor type 1 (ITPR1) in curcumin-treated U2OS cells serves an anticancer role by regulating proliferation, apoptosis, migration, and invasion [132]. Researchers concluded that 15 μM of curcumin suppressed migration and invasion; however, the cytotoxic effect induced by the same concentration of curcumin should not be neglected.

Curcumin in HOS cells
In HOS cells, curcumin induces phase arrest in G 1 /S and G 2 /M, suppressing cyclin D1, cdc2, and cyclin B1, and activates apoptotic processes through caspase 3 activation and PARP cleavage [133]. By encoding the ATP-binding cassette sub-family B member 1 gene, namely the multidrug resistance (MDR)1 gene, which is believed to be the main mechanism of MDR in human osteosarcoma cells, it could overexpress P-glycoprotein (P-gp) [134,135]. Curcumin-mediated reversal of MDR correlates with inhibition of P-gp expression and P-gp efflux pump in vitro using MNNG/HOS/MTX cells with different cross-resistance degrees to diamminedichloroplatinum, adriamycin, ifosfamide, and epirubicin, and in vivo using a xenograft-nude mouse model [136]. Although three mice bearing MNNG/HOS/MTX cells with lung metastasis were detected in the MTX group and no cases were observed in the CUR/MTX group, it is controversial to speculate about the antimetastasis abilities of curcumin before excluding its cytotoxic effect.
Researchers have shown that a mixture of curcuminoid, composed of 77% curcumin, 17% DMC, and 3% BDMC, has enhanced suppression of transforming growth factor (TNF)-stimulated NF-κB in myelogenous leukemia KBM-5 cells and the highest cytotoxicity in pancreatic cancer PANC-1 cells, followed by curcumin, DMC, and BDMC, respectively [112,137]. All curcumin, DMC, and BDMC reduce the viability of U2OS and HOS cells and increase the apoptosis induction of HOS cells by activating Smad 2/3 or inhibiting Akt; moreover, the combined use of them synergistically reduces cell viability and colony formation and induces apoptosis compared to single or two agents [138].

Curcumin in MG-63 and U2OS cells
Curcumin causes significant inhibition of cell growth and G 2 /M phase cell arrest and IC 50  Furthermore, curcumin down-regulates estrogen-related receptor (ERR)α gene expression to induce apoptotic cell death by up-regulating miR-125 in MG-63 and U2OS cells [140]. Overexpression of ERRα has reduced curcumin-induced apoptosis and ROS to confer resistance to curcumin in MG-63 and U2OS cells, while silencing of ERRα could sensitize U2OS cells to curcumin, resulting in growth inhibition and up-regulation of cleaved forms of caspase 7 and PARP.

Curcumin in MG-63 and HOS cells
miR-21 is up-regulated in osteosarcoma tissues, and its knockdown decreases the migration and invasion of MG-63 cells [141,142]. On the other hand, miR-21 inhibits apoptosis of Saos-2 cells by targeting caspase 8 [143] and can predict poor prognosis in osteosarcoma patients [144]. In MG-63 and HOS cells, curcumin up-regulates the reversion-inducing cysteine-rich protein with the Kazal motifs (RECK) protein expression by inhibiting miR-21, and thus subsequently deregulates the canonical Wnt/β-catenin signaling that leads to apoptosis of, indicating that the miR-21/RECK/Wnt/β-catenin pathway plays an influential role [145].

Curcumin in U2OS and Saos-2 cells
Curcumin exhibits an antiproliferative effect and suppresses the canonical Wnt/β-catenin pathway by repressing both extrinsic and intrinsic and activated β-catenin/T-cell factor (Tcf) transcriptional activities in U2OS, Saos-2, and HOS cells [146]. At a concentration of 5 μM, curcumin improves cytotoxicity, induction of apoptosis, and targeting mitochondria of a C-1 acetoxymethyl analog of 7-deoxypancratistatin, JC-TH-acetate-4 (JCTH-4), selectively in U2OS cells [147]. Within the cytotoxic concentration range, 5-20 μM of curcumin also decreased U2OS cell migration and reversed Wnt/β-catenin-enhanced U2OS cell invasion under inhibition of the intrinsic transcriptional activity of β-catenin/Tcf and disruption of nuclear translocation of the β-catenin protein in part by down-regulating MMP-9, a Wnt response gene [146]. The authors should, however, exclude the cytotoxic effect before concluding about the antimigratory effect. In Saos-2 cells, curcumin within 6-50 μM could inhibit cell survival and resveratrol and diallyl disulfide enhance cell apoptosis induced by curcumin [148]. In U2OS and Saos-2 cells, curcumin could increase H3K18 acetylation (H3K18Ac), which is a post-translational modification of the core histone and commonly correlates with active genes [149,150].
Overall, the authors concluded curcumin had antimigratory potential under the cytotoxic concentrations of curcumin in the same study [59,116,125,126,132,136,139,146]. Therefore, the clinician should determine whether curcumin has the antimetastatic ability to scale up to a level before applying it meaningfully and practicably.

Curcumin analogs in human osteosarcoma
By improving curcumin's solubility, absorption, and stability to increase its bioavailability and potency, this promising natural product will make its way to the forefront of human osteosarcoma therapeutic agents shortly. As such, several curcumin analogs have been further developed to increase the ability to i.v. administration and to prolong half-lives compared to their parent compound to treat osteosarcoma [151,152] (Figure 4). Through chemical modification to trigger different activities and mechanisms, these anti-osteosarcoma-targeting analogs become more soluble, stable, and potent.
In human osteosarcoma SJSA and U2OS cells, FLLL32 suppresses cell proliferation more than curcumin and enhances proteasome-mediated degradation of STAT3, resulting in a subsequent loss of vascular endothelial growth factor (VEGF), MMP-2, and survivin expressions, and induction of apoptosis [154] (Table 2). Despite inhibiting STAT3 function and expression, FLLL32 decreases STAT3 DNA binding in SJSA cells. Furthermore, the specific inhibitor of JAK2/STAT3, FLLL32, not only decreases cell growth in human osteosarcoma 143.98.2 cells but also delays tumor growth by reducing cell proliferation and inducing cell apoptosis in a mouse model of the 143.98.2 xenograft nude [155]. Furthermore, genetic inhibition of STAT3 prevented osteosarcoma 143.98.2 xenograft growth in vivo, suggesting that targeting JAK/STAT3 may be a promising therapeutic strategy for osteosarcoma patients.

2c, 2f, 3c, 3f, 4c, and 4f in human osteosarcoma
Curcumin analogs 2c, 2f, 3c, 3f, 4c, and 4f have been synthesized to reduce the β-catenin/Tcf activity to inhibit the Wnt/β-catenin pathway in human osteosarcoma U2OS cells [156]. Although 2f, 3c, and 4f have not altered the amount of β-catenin in the cytoplasm of U2OS cells, they could cause a reduction in nuclear β-catenin at 5 μM, indicating a disruption in the translocation of nuclear β-catenin. Reduced U2OS cell invasiveness was also observed at the same concentration (5 μM) and 1 μM by 2f, 3c, and 4f. However, their cytotoxic effects should be excluded before making speculations on the anti-invasive capabilities of U2OS cells to avoid misleading anti-invasiveness by cell death.

EF-24 in human osteosarcoma
To make the structure simple and symmetrical, the designated monoketone analog of curcumin 3,5-bis(2-fluorobenzylidene) piperidin-4-one, also known as EF-24, exhibits broad-spectrum antiproliferative activity in some cancer cell lines [157]. The half-life values of the absorption and elimination of EF-24 are 177 and 219 min, respectively, and its bioavailability is 60% and 35% after oral and i.p. administration, respectively. In human osteosarcoma Saos-2 cells, EF-24 has a higher potency than curcumin to inhibit cell growth and to induce cell death, change nuclear morphology, and activate caspases 3 and 7 through the Fas/PARP axis, indicating that EF-24 strongly induces apoptosis through both the death receptor-mediated extrinsic pathway and the mitochondrial-mediated intrinsic pathway [158].
EF-24 also increases levels of malonydialdehyde (MDA), ROS, and intracellular ferric ions, and these effects are attenuated by ferrostatin-1 [159]. EF24 up-regulates HO-1 expression and HO-1 overexpression facilitates EF24 to induce ferroptosis in U2OS and Saos-2 cells while silencing of HO-1 attenuates EF24-mediated cytotoxicity and inhibition of glutathione peroxidase 4 (GPX4) expression. These results of up-regulation of HO-1 by EF24 suppressing GPX4 expression and triggering ferroptosis indicate that EF24 may serve as a promising agent for the treatment of patients with HO-1-positive osteosarcoma.

DK1 in human osteosarcoma
To improve bioavailability, the synthesized chemical curcumin analog DK1, (Z)-3-hydroxy-1-(2hydroxyphenyl)-3-phenylprop-2-en-1-one, has been synthesized [161]. In addition to cytotoxic activity in breast MCF-7 cancer cells by inducing G 2 /M cell cycle arrest and apoptosis, DK1 leads to more morphological changes in human osteosarcoma U2OS and MG-63 cells and a significant reduction in cell numbers by inducing apoptosis than curcumin, which occurs through the mitochondrial-dependent signaling pathway [162]. Furthermore, DK1 has been reported to inhibit metastasis and angiogenesis by up-regulation of PI3K/Akt in U2OS cells and NF-κB in both U2OS and MG-63 cells [48]. However, the authors should exclude the effects of IC25 and IC 50 on cell growth before drawing any conclusions about the inhibition of DK1 motility, migration, invasion, and angiogenesis.

Cur C086 in human osteosarcoma
Having been artificially synthesized, the curcumin derivative C086 (cur C086) [1,4,7-tris (4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dio ne], is a structural analog of curcumin and has superior therapeutic effects than natural curcumin, such as aqueous solubility and anticancer properties in colon cancer [166]. In human osteosarcoma MG-63 cells, cur C086 down-regulates B lymphoma Mo-MLV insertion region (BMI)1 mRNA and protein expressions and inhibits proliferation dose-and time-dependently [167]. Its IC 50 value for cur C086 is 20 μM and that for cisplatin is 1.28 nM. Although the expression of the polycomb complex protein BMI1 and cell proliferation exposed to cur C086 (20 μM)+cisplatin (1.28 nM) has been decreased, they could synergistically increase the killing effect and induce apoptosis in MG-63 cells. Furthermore, cur C086 treatment increases the chemosensitivity of MG-63 cells to cisplatin or 5-FU. Using the cytotoxic concentration of 20 μM, cur C086 suppresses migration and invasion of MG-63 cells and cur C086+cisplatin (1.28 nM) decreases invasive abilities compared to the control, cur C086, and cisplatin groups. Thus, the authors could not speculate on the antimetastasis effects of cur C086 because cytotoxicity at 20 μM could mislead. Furthermore, proteins P16 and E-cadherin are markedly down-regulated and inhibition of EGFR and Notch 1 is observed in the cur C086+cisplatin treatment group compared to the single treatment group. Therefore, cur C086 is a promising chemotherapeutic agent and cur C086+cisplatin may be a potential chemotherapeutic form for osteosarcoma patients.

L48H37 in human osteosarcoma
Compared to the β-diketone structure of curcumin, the novel curcumin analog L48H37 with an unsaturated monoketone structure1-ethyl-3,5-bis[(E)-3,4,5-trimethoxybenzylidene]piperidin-4-one, was manufactured to enhance its bioavailability and antitumor properties in human lung cancer A549 cells and human pancreatic ductal adenocarcinoma PANC-1 and MIA PaCa2 cells [168,169]. Up to 5 μM in human osteosarcoma U2OS and MG-63 cells without cytotoxicity, L48H37 suppresses their motility, migration, and invasion [170]. L48H37 reduces the protein level, mRNA expression, and promoter activity of the urokinase plasminogen activator (uPA) to interfere with migratory activity in U2OS cells, and also represses the phosphorylation of STAT3, JAK1, JAK2, and JAK3. While STAT3 inhibitor C188-9 decreased uPA expression and migration potential in U2OS cells, STAT3 activator colivelin countered the decrease in uPA expression caused by L48H37, consistent with suppression of invasion and migration by inhibiting uPA expression through the JAK/STAT pathway.

GO-Y078 in human osteosarcoma
By directly interacting with the substrate binding site and its solubility to overcome poor bioavailability, (1E,4E)-1-(4-hydroxy-3,5-dimethoxyphenyl)-5-(3,4,5trimethoxyphenyl)-penta-1,4-dien-3-one (GO-Y078) was derived from curcumin to enhance inhibition of cell growth [60,171,172]. GO-Y078 reduces the viability of human osteosarcoma U2OS, MG-63, 143B, and Saos-2 cells in a dose-dependent manner and induces sub-G1 fraction cell arrest and apoptosis in U2OS and 143B cells [173]. Although GO-Y078 activated both intrinsic caspase 9 and extrinsic caspase 8 initiators to trigger the caspase 3 cascade and PARP in U2OS and 143B cells, suppression of cellular inhibitor of apoptosis (cIAP)-1 and X-chromosome-linked IAP (XIAP) could be detected. In addition to inhibiting IAPs, GO-Y078 simultaneously activates intrinsic and extrinsic apoptotic pathways and cell arrest in U2OS and 143B cells by triggering the JNK and p38 signaling pathways. These results provide a better insight into the mechanisms responsible for the apoptotic properties of GO-Y078 on human osteosarcoma cells and the recognition of GO-Y078 as a promising agent for the treatment of osteosarcoma.

Controlled Formulations for Delivery to Human Osteosarcoma
Due to poor aqueous solubility, insufficient absorption, extravagant metabolism, and hasty systemic clearance, the limited bioavailability of curcumin has hampered its clinical application [60]. To harness the anticancer abilities of curcumin, researchers have designed numerous encapsulationbased delivery systems using multifunctional lipid/ liposomes, nanoformulations (NPs, nanocarriers, nanoarrays, and hydrogels), micelles, three-dimensional (3D) printed scaffolds, and adjuvants to improve curcumin solubility and allow targeting specific cells and organs of osteosarcoma, ultimately promoting therapeutic effectiveness [113,176,177]. By applying different materials and techniques to prepare more bioavailable, targeting and penetrating osteosarcoma cells, various formulation methods and delivery systems can be classified as follows: Cur-SPNP Increases cytotoxicity, inhibition of cell growth, and intracellular ROS All ratios inhibit cell growth, with no difference in IC50 between Cur/SPNPs ratios.

Lipid particle
In 11 patients aged 12-60 years (average 18.3) with metastatic high-grade osteosarcoma (7 men and 4 women), high plasma concentrations and non-linear dose dependence could be obtained by oral formulation of solid lipid curcumin particles (SLCPs), and acceptable tolerability has been observed [1] ( Table 3). However, the increased bioavailability of curcumin by SLCP was still not defined in the study, as it could be due to increased absorption or reduced conversion of free curcumin.

Liposomal NP
The liposome, a globular shape with a central aqueous space and an outer lipid bilayer, varies from 25 to 1,000 nm in diameter [178]. It contains one phospholipid bilayer with a hydrophobic tail and a hydrophilic head in the aqueous solution. For drug delivery system targeting, hydrophilic molecules are incorporated into the aqueous interiors of the outstanding weapon of liposomes, while lipophilic particles are incorporated into their hydrocarbon bilayer. Using NP or ultrafine particle technology between 1 and 100 nm in diameter, nanoformulations have been developed to resolve the problem of poor bioavailability of the drug. By forming water-soluble complexes through double encapsulation in cyclodextrins (CDs) and followed by liposomes, 2-hydroxypropyl-γ-CD (HPγCD) enhances the water solubility of curcumin from 11 ng/mL to 600 μg/mL, about ~10 4 -fold increase in the aqueous phase of the vesicles [113]. When dissolved in dimethylsulfoxide (DMSO), curcumin has been more cytotoxic to osteosarcoma cells at all concentrations investigated [105,131,179]; however, HPγCD-curcumin liposomal NPs initiate the caspase cascade that results in apoptotic cell death compared to autophagic cell death induced by DMSO-curcumin in human osteosarcoma KHOS cells and KHOS mice [113]. The cytotoxic effects of liposomal curcumin on KHOS cells are 2 to 4 times more potent than those detected in non-liposomal formulations.
Because C6 ceramide (C6) is highly insoluble like curcumin, C6 and curcumin have been incorporated into the bilayer of liposomal NP to increase its bioavailability and cytotoxic effects against human osteosarcoma MG-63 and KHOS cells [115]. C6-curcumin liposomal NP induces G2/M arrest and exhibits combined effects on the expression levels of cyclin D1 and cyclin B1. As liposomal curcumin pegylation has generated 4-5 times more effective than curcumin as a pure compound against osteosarcoma [113], the preparation of curcumin liposomes improves the circulation half-life of curcumin for in vivo use [180]. As overexpression of the folate (FA) receptor in most cancer cells becomes more malignant, the C6-curcumin-FA liposome represses tumor size and appears to show more apoptosis in KHOS xenograft mice [115].

Amphiphilic NP (APNP)
The NP is called amphiphilic because it contains hydrophilic and hydrophobic functional groups. Using a co-dissolution method with acetic acid and dialysis tubes, a homogeneous curcumin solution has been formed in the hydrophobic core of spherical APNP to improve solubility and selectively inhibit the proliferation of human osteosarcoma MG-63 cells [179]. Compared to curcumin dissolved in DMSO, the cytotoxicity of curcumin-loaded APNPs in the concentration range of 20-30 μM is more selective for MG-63 cells. Although the viability of MG-63 cells is as low as 15% after treatment with curcumin-loaded APNP at a curcumin concentration of 30 μM, over 50% of human osteoblasts are viable under the same conditions. Therefore, the surfactant-like drug carrier that encapsulates hydrophobic curcumin in its hydrophobic core could selectively inhibit osteosarcoma cells more than healthy osteoblasts.
Poly(D,L-lactide-co-glycolic acid) (PLGA) NP Although water-soluble curcumin-loaded PLGA NP, which improves oral absorption of curcumin, exhibits antiproliferation effects in cisplatin-resistant human oral cancer CAR cells, there is little cytotoxicity for normal human oral keratinocytes and gingival fibroblasts [181]. Intriguingly, curcumin-PLGA NP triggers apoptosis of human osteosarcoma U2OS cells through the mitochondria-dependent caspase cascade and the Akt-Bad pathway by up-regulating the expression of cleaved caspases 3 and 9, Bad, cytochrome c, and Apaf-1, as well as down-regulating the expression of p-Akt [182].

Polymeric NP (PN)
Polymeric NP can circumvent the drawback of poor aqueous solubility of curcumin to improve its systemic circulation. To overcome both dose-limiting side effects and the therapeutic failure incurred by MDR in osteosarcoma, co-encapsulated lipid-coated PN (LPN) of mixed biodegradable polymer core and lipid monolayer shell has been designed to improve inhibitory cell viability of human osteosarcoma KHOS cells [183]. Without obvious toxicity, promising dual drugs DOX+Cur co-encapsulated LPNs also enhance antitumor efficiency in KHOS-bearing mice. Accordingly, drug nanotechnology exhibits more curative effects in vitro and in vivo than its free-drug counterparts. Using spherical polymeric NP as a curcumin carrier (Cur LPN) to optimize curcumin uptake, Cur LPN in HOS cells blocks tumor migration, invasion, and stemness driven by the secretome of acid-stressed mesenchymal stromal cells [184].
In addition to their small size for easy transmission into the cell with less toxicity, such as immunogenicity, and slow degradation and clearance, protein NPs can actively improve curcumin's biodegradability, stability, modification, and control [185]. By controlling the particle size and Cur/soy protein NP (SPNP) ratio, a slow-release Cur-containing SPNP (Cur-SPNP) has been designed to increase cytotoxicity, inhibitory activity on cell growth, and intracellular ROS levels in human osteosarcoma Saos-2 cells. Among IC50 values of curcumin/SPNP ratios (1:10, 1:20, 1:30, and 1:40), no difference could be detected, whereas all ratios inhibit Saos-2 cell growth. The novel structures for encapsulation and delivery of curcumin increase targeting at the site to improve therapeutic efficiency, as well as to reduce dosage and side effects [186].
Mesoporous silica NPs (MSNs), inorganic-based nanocarriers, characterize a large size with high surface area and large pores to suit high loading capacity and cancer targeting [187]. As a ligand molecule, FA may be effectively used to combine with MSNs, loaded with curcumin and a variety of drugs, to target cancer. A phosphonate-functionalized MSN has been fabricated to load colchicine (CL) and curcumin as a core and to bear a polymeric coating of chitosan (CS)-cellulose (CE) conjugated with a coating shell of FA-binding ligand MSNPCLCur/CSCEFA. The constructs MSNPCLCur and MSNPCLCur/ CSCEFA improve anticancer activity (increased p53, Bax, and caspase 3, as well as decreased Bcl-2) against human HOS cells.

Micelle
A micelle is a colloidal particle formed by the aggregation of amphiphilic molecules dispersed in a liquid. For the delivery of water-insoluble drugs to cancer cells, invertible micellar polymer nanoassemblies (IMAs) have been developed by changing the macromolecular conformation, solubilizing lipophilic drugs in the interiors of IMAs, increasing the polymer concentration, and making local changes to the environmental polarity [188]. IMA-delivered curcumin could effectively decrease cell viability in human osteosarcoma MG-63, KHOS, and LM7 cells, but did not influence normal human osteoblasts [189]. In MG-63 cells, IMA-delivered curcumin induces G2 arrest, contributing to antigrowth and increasing curcumin uptake, thus developing a unique delivery system targeting MG-63 cells.
Owing to overexpression of the CD44 HA receptor and the receptor of hyaluronan (hyaluronic acid, HA)-mediated motility (RHAMM) on the tumor cell surface, tumor cells with high metastatic activity often exhibit increased binding and uptake of HA [190]. Alendronate (ALN), a nitrogen-containing bisphosphonate, is currently used to treat bone-related diseases and can be utilized as a bone-targeting ligand [191]. A micelle formed by combining hydrophilic HA and hydrophobic octadecanoic acid with ester bonds is stable, and the amphiphilic copolymer HA-octadecanoic acid (HA-C18) has been prepared to be a stable micelle and the curcumin-loaded ALN-HA-C18 micelle exhibits a high affinity for bone and enhances cytotoxic effects on MG-63 cells [176]. In mice carrying MG-63, the curcumin-loaded ALN-HA-C18 micelles effectively delay tumor growth, suggesting that ALN-HA-C18 is a potential micelle for delivering hydrophobic curcumin and targeting osteosarcoma.

Hydrogel
With the development of MDR, the therapeutic effect of curcumin may be affected, although it is a significant obstacle faced with osteosarcoma chemotherapy [192]. β-CD was adopted to enhance the physicochemical properties of curcumin by incorporating a hydrophobic inner cavity and hydrophilic hydroxyl moieties surrounding the outer surface [193]. Compared to natural curcumin in phosphate-buffered saline, a CD-Cur inclusion complex with better solubility and stability has been generated to exhibit significant cytotoxicity efficiency in human osteosarcoma Saos-2 cells [114]. Furthermore, the poly(D,L-lactide-co-glycolide)poly(ethylene-glycol)-poly(D,L-lactide-co-glycolide) thermosensitive hydrogel has been selected and synthesized as a novel nanodrug delivery system (NDDS) to co-deliver CD-Cur and DOX to tumor sites of osteosarcoma.
Gel+DOX+CD-Cur could down-regulate Bcl2, which is closely associated with the prognosis of osteosarcoma patients [194], and up-regulate the protein level of caspase 3, indicating that dual drug nanotechnology improves cytotoxicity consequences and promotes the pro-apoptotic effect of DOX and curcumin bioavailability [114].
Photopolymerization of hydrogels and water-rich 3D structures of target tissue provides mechanical stability and space for tissue regeneration to engineer tumor microenvironments [195,196]. HA is esterified in methacrylate (MA) hydrogel and attracts researchers because of its biological activities and low immunogenicity [197]. Silk fibroin (SF), which is extracted from the silkworm (Bombyx mori) cocoon, is widely applied in hydrogel preparation owing to its high biocompatibility, distinctive mechanical characteristics, and a controllable biodegradation rate [198]. The development of a HAMA/SF composite hydrogel further enhances its superior mechanical and biocompatible bioactivity to improve tissue reconstruction. For osteosarcoma therapy and bone regeneration, the curcumin-loaded chitosan NP encapsulated SF/HA esterified by MA (CCNPs-SF/HAMA) hydrogel has been developed by treating ethanol and photocuring to provide long-term treatment of osteosarcoma [199]. CCNPs-SF/HAMA hydrogel may offer a more sustainable drug release that has an equivalent curcumin concentration of 150 μg/mL for osteoblast proliferation to repair bone defects.

Adjuvant
Because of Ti's excellent potential for biocompatibility, mechanical properties, and corrosion resistance, Ti implants have been increasingly used in recent decades. The plasma spray technique is used to form a homogeneous hydroxyapatite (Ha) coating on the surface of the Ti implant to enhance osseointegration at the tissue-implant interface [200]. Due to the stimulation of bone formation and reduction of vitamin K bone resorption [201], the dual-drug delivery of curcumin and vitamin K 2 (Cur+Vit K 2 ) from plasma-sprayed Ha-coated Ti implants enhances osteoblast attachment and proliferation in vitro, inhibiting the proliferation of human osteosarcoma MG-63 cells, and in vivo osseointegration. The results also highlight the potential of Cur+Vit K2-loaded Ha-coated Ti implants for chemoprevention and postoperative defect repair in the treatment of osteosarcoma [202].

Nanocarrier
The localized NDDS directly targets cancer cells to enhance the efficacy of the hydrophobic drug, and calcium phosphate (CaP) nanocarriers are an ideal carrier for any local bone treatment [203]. Among the CaP nanocarriers, the carbonated apatite (CA) nanocarrier closely mimics natural bone minerals and shows superior suitability as a curcumin carrier compared to the Ha nanocarrier because of its lower crystallinity, increased reactivity, and increased solubility [204]. Eggshells mainly comprise calcium carbonate, and eggshell-based apatites exhibit higher levels of curcumin loading and release. The curcumin-loaded eggshell-derived CA nanocarrier exhibits high anti-inflammatory activity and has high cytotoxicity against human osteosarcoma MG-63 cells. These results suggest that eggshell-derived apatite drug nanotechnology is a very suitable way to cure osteosarcoma, prevent post-cancer inflammation, and modulate bone repair and regeneration.
Guided by an external magnetic field, superparamagnetic iron oxide NP (SPION), the most common biocompatible magnetic core, can magnetically control tumor-targeting drug delivery and a switchable hyperthermia system [205]. To solve the issue of side effects, chemotherapy resistance, and limited survival ratio, a magnetothermal nanocarrier containing SPIONs coated with a combination formulation of a 3-block copolymer Pluronic F127 and F68 on oleic acid and curcumin-loaded (SPION@OA-F127/F68-Cur) has been designed [206]. The nanocarrier SPION@OA-F127/F68-Cur controls the gelation temperature of the shell and the release of curcumin to synergistically exhibit promising bifunctional therapeutic potentials of chemo-co-thermal efficacy in killing human osteosarcoma MG-63 cells.

Nanoarray
To overcome postoperative osteosarcoma recurrence during bone regeneration, the Ti-based nanoarray device has been utilized for its analogous elasticity to natural bones and has served as a drug vehicle for various therapeutic agents [207]. The curcumin-loaded Ti dioxide (TiO2) nanoarray has been manufactured using functional CD-based polymer (pCD) coatings and a polydopamine (pDA)-assisted film implemented as the first coating layer on the surface of the TiO 2 nanoarrays to possess both cancer cell killing ability and excellent bioactivity [208]. Curcumin modifies the pCD functionalized TiO 2 /pDA surface to create TiO 2 /pDA/pCD/Cur and promotes apoptosis of human osteosarcoma MG-63 cells by inducing mitochondrial dysfunction caused by ROS overproduction, and, at the same time, inhibits tumor growth of mouse-derived UMR-106 osteosarcoma cell xenograft models. These results successfully demonstrate that the as-prepared TiO2/pDA/pCD/Cur construct has anticancer performance and high biocompatibility.

Nanofibrous scaffold
Bone scaffolds have a superior potential to repair bone defects after surgical excision by providing better physical support, nutrients and waste transportation, and new tissue ingrowth [209]. Encapsulation of curcumin in a liposome to increase bioavailability, followed by incorporation into 3D printed CaP scaffolds with designed porosity, facilitates patient-specific implant fabrication with mechanical interlocking between surrounding host tissue and scaffold [210]. Additionally, liposomal curcumin promotes the attachment, proliferation, viability, and differentiation of human fetal osteoblasts. Intriguingly, a Cur-encapsulated liposomecoated 3D printed scaffold with bifunctional bone tissue engineering inhibits human osteosarcoma MG-63 cells and also promotes the formation of healthy bone cells within the porous scaffold, offering a promising drug delivery strategy for the treatment of bone defects after tumor resection [177].

Conclusions and perspectives
To improve poor bioavailability, several current curcumin analogs offer better absorption, stronger potency, slow metabolism, and delayed elimination ( Figure 5). To enhance therapeutic efficacy, multifunctional nanoplatforms, such as liposomes, NPs, nanocarriers and nanoarrays, micelles, 3D printing, and other promising novel formulations provide longer periods of circulation, resistance to the metabolic process, and improved targeting of curcumin. However, the existing literature does not yet provide enough evidence to conclude that curcumin in controlled formulations and its analogs are effective in treating human osteosarcoma. Despite combining the advantages of material and design, there is no so-called "most effective" design to co-deliver curcumin and other medicines because the ideal development is in progress; better analogs and carrier systems will be required in the future to achieve better results. In addition to future clinical trials, serious study designs to elucidate and clarify the antimetastatic issue of curcuminoids are warranted to offer more efficient strategies for patients with osteosarcoma.